Tracking Single Proteins within Cells

We present experiments in which single proteins were imaged and tracked within mammalian cells. Single proteins of R-phycoerythrin (RPE) were imaged by epifluorescence microscopy in the nucleoplasm and cytoplasm at 71 frames/s. We acquired two-dimensional trajectories of proteins (corresponding to the projection of three-dimensional trajectories onto the plane of focus) for an average of 17 frames in the cytoplasm and 16 frames in the nucleus. Diffusion constants were determined from linear fits to the mean square displacement and from the mean displacement squared per frame. We find that the distribution of diffusion constants for RPE within cells is broader than the distributions obtained from RPE in a glycerol solution, from a Monte Carlo simulation, and from the theoretical distribution for simple diffusion. This suggests that on the time scales of our measurements, the motion of single RPE proteins in the cytoplasm and nucleoplasm cannot be modeled by simple diffusion with a unique diffusion constant. Our results demonstrate that it is possible to follow the motion of single proteins within cells and that the technique of single molecule tracking can be used to probe the dynamics of intracellular macromolecules.